Electroporation of HAP1, K562, and U2OS cells

HAP1 cells were electroporated using the SE Cell Line 4D-Nucleofector X Kit S (Lonza) according to the manufacturer’s protocol with 4 × 10⁵ cells (program DZ-113), 300 ng PE2–P2A–BSD, 100 ng pegRNA plasmid, and 33 ng sgRNA plasmid (where indicated). After electroporation, cells were cultured in 48-well plates (Corning) with IMDM plus GlutaMAX supplemented with 10% FBS. The day after electroporation, media was replaced with fresh IMDM plus GlutaMAX supplemented with 10% FBS and 10 ng μL^-1 blasticidin (Thermo Fisher Scientific) to select for cells expressing prime editor.

K562 cells were electroporated using the SF Cell Line 4D-Nucleofector X Kit S (Lonza) according to the manufacturer’s protocol with 5 × 10⁵ cells (program FF-120), 800 ng prime editor plasmid, 200 ng pegRNA plasmid, 83 ng sgRNA plasmid (where indicated), and 400 ng MLH1dn plasmid (where indicated). After electroporation, cells were cultured in 6-well plates (Corning) with RPMI 1640 medium supplemented with 10% FBS and 292 μg mL^-1 L-Glutamine (Corning).

U2OS cells were electroporated using the SE Cell Line 4D-Nucleofector X Kit S (Lonza) according to the manufacturer’s protocol with 2 × 10⁵ cells (program DN-100), 1600 ng PE2 or PE2–P2A–MLH1dn plasmid, 400 ng pegRNA plasmid, and 166 ng sgRNA plasmid (where indicated). After electroporation, cells were cultured in 12-well or 24-well plates (Greiner Bio-One) with McCoy’s 5A medium supplemented with 10% FBS.